In the brain, many pathophysiological events are accompanied by acidification of the extracellular environment. Unsurprisingly, neurons often show robust electrophysiological responses during such changes in extracellular pH. Acid sensing ion channels (ASICs) mediate the vast majority of these currents and the last few years have seen a substantial increase in our understanding of ASIC neurobiology. Because of this research, ASICs have been implicated in cell death following stroke, in pain, in fear related memory and in drug addiction amongst other disorders. However, we have not seen a corresponding surge in our understanding of how ASICs operate at a molecular level. Without such knowledge, we cannot translate our growing awareness of neurobiology into useful treatments. The candidate will combine his expertise in electrophysiology and fluorescence with new training in cryo-electron microscopy (cryo-EM) to bridge the gap between ASIC neurobiology and biophysics. This will be done through three Aims. During Specific Aim 1 in the K phase, the candidate will obtain high resolution cryo-EM structures of ASIC1a apo and desensitized states. New training in protein purification will be provided from Dr. Jayaraman and ASIC-specific advice from Dr. Gonzales. Dr. Serysheva will provide expert instruction in cryo-EM imaging and 3D reconstruction to a level where the candidate can perform the experiments independently. These structures will provide the first glimpse of the apo state and determine if steady-state desensitization (provoked by pH's near 7) is the same conformation as desensitization following activation (pH's near 5). The R phase will begin with Aim 2 where the candidate and his trainees will use concatenated subunits to determine the stoichiometry of activation and establish if conformational changes are concerted across subunits. In Aim 3, the candidate and his trainees will obtain high resolution cryo-EM images of heteromeric ASICs, the predominant population in the brain. This combination of functional information and structural data will help fulfill the candidates two near term goals of contributing essential insight into th fundamental operation of ASICs and emerging as a leader in the field of ASIC gating. Moreover, these studies will provide preliminary data to develop a competitive R01 application focusing on ASIC heteromeric structure and function. This K99/R00 award will also better position the candidate to satisfy the long term goals of rendering the effect of ASIC mutations or manipulations predictable and to help drive efforts to develop more selective and useful pharmacological agents.